RAPID NUCLEAR TRANSLOCATION AND INCREASED ACTIVITY OF CYCLIN-DEPENDENT KINASE-6 AFTER T-CELL ACTIVATION

To elucidate the roles of cyclin-dependent kinase 6 (cdk6) in T cells, we examined its intracellular localization, kinase activity, and asso ciated proteins in the jurkat T lymphoblastoid cell line, jurkat cells had a high level of cdk6, which was associated with cyclin D3, but no t cyclin D2, the member of the cyclin D family. When stimulated by a c ombination of PHA and anti-CD28 mAb, cdk6 activity was up-regulated, a s measured by an in vitro kinase assay using recombinant, truncated re tinoblastoma tumor suppressor gene protein (Rb protein) as substrate. Activation was most prominent when cells were stimulated with the comb ination of PHA and anti-CD28, although significant increases were dete cted after stimulation with PHA alone. The combination also resulted i n maximal activation of c-Jun kinase and IL-2 production. Costimulatio n resulted in a rapid translocation of cdk6 to the nucleus, as demonst rated by both confocal immunofluorescence microscopy and biochemical f ractionation techniques. Cdk6 activation and nuclear translocation wer e also observed after stimulation of Jurkat cells using the anti-CD28 Ab in combination with a mAb to CD3 (OKT3). Furthermore, nuclear trans location was observed in normal human T lymphocytes isolated from peri pheral blood and stimulated in vitro with PHA. Two potential endogenou s cdk6 substrates (with apparent molecular masses of 75-80 and 55-60 k Da), which were immunoprecipitated with cdk6 and phosphorylated in the in vitro kinase assay, were also identified. These data demonstrate t he rapid activation and intracellular translocation of cdk6, implicati ng this kinase in early signal transduction events in T cells.